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This paper describes an electrochemical immunosensor for the detection of target molecules without the use of labeled antibodies. The sensor is based on the combination of a gold electrode covered with a conductive self-assembled monolayer (SAM) of mixed thiols and a permeability measurement of an antibody layer anchored on the SAM. The permeability was measured by the square wave voltammetry (SWV) of ferrocyanide added in a measurement solution. The conductive SAM allows constant electron transfer between the electrode and ferrocyanide, providing a stable signal during repeated SWV measurements in contrast to immunosensors using conventional SAM techniques. We demonstrate that our sensor using the Anti-human chorionic gonadotropin (hCG) antibody selectively detects hCG in a minute without any washing process. The presented label-free immunosensor technology is fundamentally suited for easy-to-use assays for biological molecules such as hormones and drugs.